Molecular Characterization And Correlation With Fruit Symptoms Of The Viroids Infecting Apple

In recent years, symptoms including mosaic, yellow spots, chlorotic ringspots, and scar skins were easily observed on apple fruits in apple-cultivated areas in China, and the symptoms have lead to a remarkable deduction of the market costs for the infected fruits. To clarify whether the symptoms were caused by viroids and their characterization, 36 apple fruit and leave samples were collected from Shandong, Shanxi and Liaoning provinces, subjected to RT-PCR detection of the four viroids infecting apple trees, and deeply characterized the molecular features of the viroids that were detected. The major results of the study are listed as follows:1. Thirty fruit or leaf samples were collected from apple trees(Malus domestica var. Fuji) showing the above mentioned fruit symptoms from Shandong and Shanxi in China, and subjected to RT-PCR detection of the four viroids infecting apple trees including Apple scar skin viroid(ASSVd), Pear blister canker viroid(PBCVd), Apple dapple fruit viroid(ADFVd), and Hop stunt viroid(HSVd). The results showed that ten samples were positive for ASSVd infection, five for PBCVd, and three for ADFVd, while no samples were infected by HSVd. Of which, two were co-infected by ASSVd and PBCVd. Correlation analysis of the varied symptoms with the infected viroids revealed that the samples showing yellow spots on the fruits were totally infected by ASSVd. Together with previous reports, the yellow spot symptoms was concluded to be induced by ASSVd, while other symptoms had no close relationship with the viroid infection. In addition, six branch samples of six different apple species were collected from Liaoning province, and subjected to RT-PCR of ASSVd and PBCVd. The results showed that three samples were infected by ASSVd, whereas no samples were infected by PBCVd.2. Four to nine clones of each of eleven ASSVd isolates were randomly sequenced, and 71 clone sequences were obtained. The sequence lengths ranged from 328 to 333 nt except that two clones of isolate HJGL from Liaoning showed abnormal sizes of 290 and 291 nt, respectively. Multiple nucleotide alignment revealed that the similarities ranged from 92.4 to 100% among the clones within the each isolate, while 93.9 to 100% among the different isolates, which was performed by aligning their consensus sequences deduced from the sequenced clone sequences within an isolate as their representative sequences.3. Molecular variation in 10 base positions and phylogenetic tree analysis of the obtained isolates showed that these isolates were be divided into two groups(groupⅠand Ⅱ). Of which, the groupⅠcontained the isolates from different regions: the six isolates obtained in this study were collected from Shandong, Shanxi and Liaoning provinces, while the others involved in this group were reported to be collected from Xinjiang and Liaoning provinces of China, India, South Korea, Canada and Iran; the group Ⅱ contained six isolates totally from China. The results showed that the groupⅡmight be an unique group composed only by the ASSVd isolates from China. The population structural analysis of the two groups exhibited the genetic relationship among the different isolates.4. Based on the consensus sequence of the 71 clone sequences, the secondary structure was predicted for the obtained ASSVd isolates, and variation sites observed for the total clones were marked along the structure. It revealed that the variation sites distributed in all the structural domains including Terminal left(TL), Pathogenicity(P), Central(C), Variable(V) and Terminal right(TR), of which a rare mutation(only one mutation) occurred in TR. If only the consensus mutations were considered within each of the 11 isolates, a remarkably lower molecular variation was observed in the structural domains, with ten mutations mainly distributing in TL, P and C domains. Of which, the mutations located in the left of TL, right of C and surrounding the P domain.5. Deep sequencing ASSVd-infected apple leaves of sample YT-1 indicated that the sizes of small interference RNAs(si RNAs) derived from ASSVd were majorly 21, 22 and 24 nt long. The si RNAs derived from plus and minus strands could cover the whole ASSVd genome in their corresponding strands, almost with equal amount but asymmetrically distributing along the genome, and both ranged to the highest distributing levels with the 21nt-sized si RNA. Two and three hotspots of si RNAs could be observed distributing over the plus and minus strands, respectively. Analysis of the 5’-terminal nucleotides of the both stranded si RNAs revealed an obvious nucleotide bias, with a dominant C nucleotide for the both strands, a tendency of C>A>G>U for the plus while C>A>U>G for the minus.